**2.2 Factors that can affect white wine protein stability**

The denaturation of some white wine proteins could result in aggregation and flocculation and sometimes in the development of deposits [25], other wine nonproteinaceous compounds can also be related to the wine protein haze development. Curiously, wines with an identical protein fraction can present different haze tendencies [41], and the wine ethanol level did not influence wine protein instability [42, 43]. The protein-polyphenol interaction is the major studied mechanism associated with white wine protein instability [44, 45]. The existence of procyanidins is necessary to develop wine turbidity, only the presence of wine proteins did not develop wine turbidity [46]. It was shown that the interaction between haze-active polyphenol and haze-active protein and the amount of haze formed is highly dependent on protein and polyphenol concentration and their ratio [47]. The turbidity of a protein-polyphenol complex increased with a pH rise from 2.5 to 3.7 (model wine solution with 10% ethanol) [48]. Some authors consider that protein haze formation is an isoelectric precipitation mechanism [49]. Some authors think that turbidity formed in white wines is related to hydrophobic interactions among proteins and tannins happening on the hydrophobic tannin-binding sites of proteins that can be

exposed depending on heating and reduction [37]. Many phenolic compounds were detected in protein haze, such as tyrosol, *trans*-*p*-coumaric, vanillic, *trans*-caffeic, protocatechuic, gallic, syringic, ferulic, shikimic acids, (+)-catechin and ethyl coumaric acid ester; quercetin and cyanidin, after acid hydrolysis, the existence of procyanidins was also shown [39]. Phenolic compounds can increase haze formation by cross-linking denatured proteins provoking aggregate development [9], in fact, the removal of phenolic compounds from wines resulted in reducing haze development [38]. The X factors are factors essential for protein turbidity and are wine conditions such as pH, ionic strength, organic acid concentration [49], polysaccharides [50], metal ions [51] polyphenols/phenolic compounds [25] and sulphate anions. As mentioned before, wine pH is an important factor in protein haze development, with model wines at pH 4.0 inducing higher protein aggregation and turbidity development after heating than model wines of lower pH (pH 3.0) [52]. The application of sulphate anions or sodium cations that increase the wine electrical conductivity and ionic strength increases the tendency of haze formation after heating, by the decrease of the electrostatic repulsion of proteins [37]. In model wines, it was shown that other ions including tartrate, chloride, Fe2+/3+ and Cu+/2+, do not influence the turbidity formation [30]. Higher electrical conductivity (0.134 and 0.163 S/m) and protein levels (9 and 25 mg/L) provoke greater perceptible turbidity; however, the white wine with low iron levels (0.3 and 0.9 mg/L) and protein stability appears to increase so there is a negative correlation between wine turbidity and the iron levels [53]. There is evidence that polysaccharides could potentially decrease wine protein instability by forming a protective layer around unfolded proteins [54]. Organic acids could present interactions with phenolic acids, free amino acids, tannins, pectic compounds and sulphate ions, avoiding in this manner, their interaction with proteins [55]. The same authors verified that organic acids could influence wine protein instability by the electrostatic interactions that depend on the organic acid pKa and protein isoelectric point values and the medium pH. The sulphate ions could be a non-proteinaceous factor for protein instability, as they promote protein-protein hydrophobic interactions Pocock et al. [38], in addition to the suppression of the electrostatic repulsion between proteins by the increase of the ionic strength of the medium [37]. It was demonstrated that potassium hydrogen sulphate can influence haze formation [7]. Some authors suggest a three-stage process in the protein haze formation that included protein unfolding, protein self-aggregation and aggregate cross-linking, highlighting the role of sulphate ions in all stages [9]. Chagas et al. [56] verified the influence of sulphur dioxide, existing in wines in the irreversible denaturation and aggregation phenomena of thaumatin-like proteins and their influence on wine protein instability or turbidity development. The presence of ion bisulphite (HSO3 − ) results in cleavage of the disulphide bonds of the thaumatin-like proteins, with the formation of S-thiosulfanates and free thiol-groups that contribute to the temperature-induced protein unfolding. The hydrophobic surfaces and the presence of free thiol-groups result in protein aggregation by formation of inter-protein disulphide bonds in thaumatin-like proteins, following a nucleation-growth kinetic mode.
